1. Field of the Invention
The present invention relates to the identification and substantial purification of a factor (INHIB) that inhibits the immunologic activity of cytokine-stimulated leukocytes. This factor is shown to be the free .beta. chain of complement C3.
The invention additionally relates to processes for preparing the INHIB molecule, methods of purifying the molecule, screening assays for the molecule, compositions containing INHIB, and uses for the molecule.
2. Description of the Related Art
Levels of circulating eosinophils and other types of leukocytes are elevated in conditions such as allergy, asthma, parasitic diseases, autoimmune disorders such as connective tissue disease, in certain types of cancer, and in patients undergoing therapy with interleukin-2 (IL-2) or granulocyte-macrophage colony-stimulating factor. Leukocytes, in particular eosinophils, persist in the circulation with a half-life of several hours to two days and then either enter the marginated (blood vessel wall) pool or infiltrate tissues. At the site of inflammation in tissue, eosinophils and neutrophils, for example, secrete toxic substances that may kill infectious organisms but also may damage host (patient) tissues. For example, in the condition known as idiopathic hypereosinophilic syndrome, eosinophil cytotoxins cause ultrastructural damage and progressive fibrosis in the major organ systems, and ultimately lead to death due to cardiac or respiratory arrest, or liver failure.
Current treatment of leukocyte-mediated inflammatory disease consists of administering a variety of anti-inflammatory, antiproliferative, or cytotoxic drugs including prednisone, vincristine, and hydroxyurea. The efficacy of these drugs varies, depending on the disease state but there is no good treatment for idiopathic hypereosinophilic syndrome, for example. All of the drugs currently used therapeutically can have severe side effects on the subject. Similarly, there is an absence of effective drug treatments for many types of inflammatory diseases (such as rheumatoid arthritis) which involve inflammatory cells other than eosinophils.
Several research groups have identified substances from biological sources that have inhibitory activity in vitro in assays that may correlate with processes in inflammation (processes that do not involve eosinophil function). These purified substances presumably may have some efficacy in the treatment of inflammatory disease; for example, uromodulin (Muchmore et al., Science 229:479 (1985), J. Biol. Chem. 259:13404 (1984), and 263:5418 (1988)), and undefined activities that protect L929 tumor cells from the toxic effects of tumor necrosis factor (TNF) (Seckinger et al., J. Exp. Med. 167:1511 (1988) and Lin et al., Immunology 63:663 (1988)). Uromodulin, for example, binds with high affinity to interleukin-1 (IL-1) and to TNF, suggesting that its activity is specific for cellular events mediated by these two factors.
It is also possible that the processing of biological fluids may produce substances with apparent anti-inflammatory activities (Justement et. al., J. Nat. Cancer Inst. 73:469). This group showed that chromatography of low density and very low density lipoproteins can oxidize the lipid moieties of these molecules, producing a molecule that inhibits monocyte cytotoxic function.
Abundant evidence demonstrates that eosinophil cytotoxic function can be enhanced by a number of immunological mediators, but there was no previous evidence of an immunological mechanism to suppress this function.
Current treatment modalities for inflammatory disease are very unsatisfactory. Any means capable of attenuating or inhibiting cellular mechanisms of inflammation would, therefore, be highly desirable for subjects suffering from certain inflammatory disease processes.